Evaluation of the usefulness of the immunoradiometric method for post-mortem measurement of insulin concentration in the intraocular fluid: preliminary results.

INTRODUCTION: In medicolegal practice, rare cases associated with suicidal, criminal or accidental overdose of insulin are both analytically and forensically challenging. The determination of insulin in post-mortem blood has limited diagnostic value, mainly on account of post-mortem chemical degradation processes (particularly hemolysis), and hence is not useful in medicolegal practice. AIM OF THE STUDY: The aims of the study were: 1) to assess the usefulness of the immunoradiometric method, used in clinical practice, for post-mortem measurement of insulin concentration in the intraocular fluid, 2) to preliminarily evaluate the usefulness of the obtained results for toxicological and medicolegal assessment, and 3) to verify on the basis of our own material the validity of the cited literature data on the effect of hemolysis on the result of insulin level determination in post-mortem blood. MATERIAL AND METHODS: The study material consisted of 93 samples of intraocular fluid collected during consecutive medicolegal autopsies performed in the Department of Forensic Medicine and Forensic Toxicology, Medical University of Silesia in Katowice. In addition, 10 samples of peripheral blood taken from living people (5 women and 5 men) for clinical and diagnostic purposes were analyzed. Insulin(e) IRMA KIT from IMMUNOTECH was used for the analyses. RESULTS: In 86 (92.5%) samples, the insulin concentration was below the analytical sensitivity of the method (less than 0.5 µIU/ml), while in 7 cases it was in the range of 1.42-24.42 µIU/ml. CONCLUSIONS: Determination of the insulin level in the intraocular fluid by the immunoradiometric method provides an opportunity for objective verification of poisoning, however this claim requires further research.

IS THERE A ROLE OF ACTH IN INCREASED CHOROIDAL THICKNESS IN CUSHING SYNDROME?

PURPOSE: To evaluate choroidal thickness (CT) in patients with Cushing syndrome (CS) with enhanced depth imaging optical coherence tomography. METHODS: Twenty-eight patients with CS and 38 healthy volunteers were enrolled in this observational cross-sectional study. Patients with newly diagnosed CS who have been admitted to Erciyes University Department of Endocrinology in 3 years time interval were compared with age- and sex-matched healthy volunteers. Choroidal thickness was measured at the fovea and 2 points nasal and 2 points temporal to the fovea with 500-µm intervals each. RESULTS: Choroidal thickness measurements were higher in patients with CS than in the control group at all examination points; however, the difference was found to be significant at the center of the fovea (367.8 ± 94.4 µm vs. 329 ± 90.5 µm) and 1,000 µm temporal to the fovea. Choroidal thickness measurements were significantly higher in adrenocorticotrophic hormone (ACTH)-dependent CS group than in the control group at all measurement points (CT at fovea 388.2 ± 92.4 µm vs. 329.1 ± 90.5 µm). All CT measurements were found to be correlated with ACTH levels. CONCLUSION: Cushing syndrome is associated with increased CT. The ACTH-dependent CS may increase CT more than ACTH-independent CS. This effect may be directly related to ACTH itself or increased plasma cortisol levels or both.

Antioxidant status and sex hormones in women with complex endometrial hyperplasia.

Endometrial tissue is under a strong influence of sex hormones. These hormones are considered as developmental factors of endometrial hyperplasia and endometrial cancer. We examined the influence of gonadotropins (follicle-stimulating and luteinizing hormone) and sex hormones (estradiol, progesterone) on oxidant/antioxidant parameters in blood and endometrial tissue of women with complex endometrial hyperplasia. In blood, superoxide dismutase activity was significantly higher in luteal phase and postmenopause compared to the follicular phase. A significant phase-related difference of glutathione peroxidase and glutathione reductase activity was recorded in the endometrium. Both enzymes had lower activity in luteal phase and postmenopause compared to the follicular phase. The linear regression analysis of individual hormonal variables against antioxidant parameters showed negative correlation between glutathione peroxidase activity and gonadotropin concentrations in the endometrium. The regression of hyperplastic to normal endometrium is the purpose of conservative treatment based on administration of progestogens or gonadotropin-releasing hormone analogues. Our findings indicate that gonadotropins influence the antioxidant enzymes activity in women with complex endometrial hyperplasia, which may affect disease development. Further studies are needed to clarify the molecular basis of hormone action on antioxidant system that may potentially initiate a development of treatments based on redox-dependent mechanism.

Prevalence and Relationships of Iron Deficiency Anemia with Blood Cadmium and Vitamin D Levels in Korean Women.

Anemia, iron deficiency (ID), and iron deficiency anemia (IDA) are common disorders. This study was undertaken to determine the prevalence of anemia, ID, and IDA in Korean females. We examined the associations between IDA, heavy metals in blood, vitamin D level and nutritional intakes. The study was performed using on data collected from 10,169 women (aged ≥ 10 yr), including 1,232 with anemia, 2,030 with ID, and 690 with IDA during the fifth Korea National Health and Nutrition Examination Survey (KNHANES V; 2010-2012). Prevalence and 95% confidence intervals were calculated, and path analysis was performed to identify a multivariate regression model incorporating IDA, heavy metals in blood, vitamin D level, and nutritional intakes. The overall prevalence of anemia, ID and IDA was 12.4%, 23.11%, and 7.7%, respectively. ID and IDA were more prevalent among adolescents (aged 15-18 yr; 36.5% for ID; 10.7% for IDA) and women aged 19-49 yr (32.7% for ID; 11.3% for IDA). The proposed path model showed that IDA was associated with an elevated cadmium level after adjusting for age and body mass index (ß=0.46, P<0.001). Vitamin D levels were found to affect IDA negatively (ß=-0.002, P<0.001). This study shows that the prevalence of anemia, ID, and IDA are relatively high in late adolescents and women of reproductive age. Path analysis showed that depressed vitamin D levels increase the risk of IDA, and that IDA increases cadmium concentrations in blood. Our findings indicate that systematic health surveillance systems including educational campaigns and well-balanced nutrition are needed to control anemia, ID, and IDA.

Evaluation of the immunoradiometric and electrochemiluminescence method for the measurement of serum insulin in children.

Human insulin is a polypeptide hormone produced, stored, and secreted by the ß-cells in the pancreatic islets of Langerhans. Its secretion is stimulated by an increase of the glucose concentration in circulation. Non-radioactive assays are frequently used in many laboratories to measure hormone concentrations, as an alternative to the traditional "gold standard" radioimmuno- and immunoradiometric assays. The precise and reliable determination of the insulin concentration is an important concern in numerous diagnostic procedures. The aim of this study was to compare two commercially available assays (manual and automated) for measurement of serum insulin concentrations. Aliquots of the 86 randomly selected serum samples were measured by Elecsys Insulin Assay (cobas e411 immunoassay analyzer, Roche Diagnostics GmbH, Mannheim, Germany) and DIAsource INS-IRMA Kit (DIAsource ImmunoAssays S.A., Louvain-la-Neuve, Belgium). Compared assays exhibit good correlation (r = 0.996). Insulin concentrations were on average 4.2 µ IU/mL lower (p < 0.05) with the cobas e411 immunoassay analyzer when compared to those measured with DIAsouce Immunoassay. Our findings suggest that electrochemiluminescence method on the cobas e411 analyzer and manual IRMA method offered by the DIAsource for the serum insulin determination could be considered interchangeable.

High lipoprotein(a) concentrations are associated with impaired endothelial function in children.

OBJECTIVE: To examine the association between familial high lipoprotein(a), or Lp(a), concentrations and endothelial function in children participating in the Special Turku Coronary Risk Factor Intervention Project study. STUDY DESIGN: Seven-month-old children (n = 1062) with their families were randomized to a risk intervention group or to a control group. The intervention group received individualized dietary counseling to reduce the total cholesterol concentration. Children's Lp(a) and lipid values were measured repeatedly. At age 11 years, children were recruited to an ultrasound study of the flow-mediated dilation (FMD) of the brachial artery. The association between relative peak FMD and Lp(a) concentration was examined in 198 control and 193 intervention group children by linear regression analyses adjusted for sex, total cholesterol concentration, and basal artery diameter. The analyses were made in both the control and intervention groups and in the familial risk children who had a parent with Lp(a) concentration greater than 250 mg/l. RESULTS: Lp(a) concentrations were similar at age 11 years in the intervention and control groups. In all control children, FMD (%) associated inversely with Lp(a) concentration: (ß [%/1000 mg/L] = -3.74, 95% CI [-6.43, -1.45]; P = .007) and in 68 familial risk children (ß = -4.92, 95% CI [-8.18, -1.66]; P = .0037). In the intervention group the associations were lacking (P > .5), and FMD in the children with high Lp(a) concentrations (>500 mg/L, n = 12) had no attenuation (P = .027). CONCLUSIONS: Familial high Lp(a) concentration is associated with attenuated endothelial function. This association may be mitigated by an early lifestyle intervention. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00223600.

[Analysis of laboratory data of 155 patients with pheochromocytoma-paraganglioma syndrome diagnosed during the past 20 years]. / A laboratóriumi diagnosztika eredményei az elmúlt 20 évben kórismézett 155 phaeochromocytoma/paraganglioma szindrómás beteg adatainak elemzése alapján.

INTRODUCTION: Laboratory diagnosis of pheochromocytoma-paraganglioma syndrome has been markedly improved during the past two decades. AIM: Retrospective assessment of diagnostic utility of urinary catecholamines and their metabolites as well as serum chromogranin A in 155 patients diagnosed at the 2nd Department of Medicine, Semmelweis University. METHOD: Urinary catecholamines and metabolites were measured using high-performance liquid chromatography with electrochemical detection in 155 patients with pheochromocytoma-paraganglioma (of whom 28.4% had hereditary background) and in 170 non-pheochromocytoma patients used as controls. Serum chromogranin A was measured by immunoradiometry. RESULTS: Sensitivity (93.2%) and specificity (87.0%) of urinary fractionated metanephrines were higher than those of urinary catecholamines (90.9% vs. 85.7%, respectively) and serum chromogranin A (88.7% and 77.5%, respectively). Urinary normetanephrine and serum chromogranin A correlated positively with tumor size (r = 0.552, p<0.0001 and r = 0.618, p<0.0001, respectively). CONCLUSIONS: These data confirm the diagnostic utility of urinary catecholamines and their metabolites. Urinary normetanephrine and serum chromogranin A may help to estimate tumour mass and probably tumour progression.

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients.

BACKGROUND: Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence. METHODS: In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni. RESULTS: Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro. CONCLUSION: We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients. GENERAL SIGNIFICANCE: This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.

The serum level of brain natriuretic peptide increases in severe subarachnoid hemorrhage thereby reflecting an increase in both cardiac preload and afterload.

BACKGROUND: The increase of serum brain natriuretic peptide (sBNP) is well known in patients with severe subarachnoid hemorrhage (SAH). However, the pathophysiology between the clinical severity of SAH and the sBNP secretion is still not clear. The aim of this study is thus to clarify the cardiovascular pathophysiological mechanisms of sBNP secretion in severe SAH patients. METHODS: From the database of multicenter prospective study (SAH PiCCO study), sBNP level was compared among initial Hunt and Kosnik (H-K) gradings on the first day. Receiver operating characteristics (ROC) analysis was applied to decide the threshold existing between severe (H-K grade 4-5) and non-severe (H-K grade 2-3) patients. Cardiopulmonary parameters were also measured with thermodilution techniques and compared between low and high sBNP groups. RESULTS: sBNP level was significantly higher in severe patients than in non-severe patients (566.5 ± 204.2 vs. 155.7 ± 32.8 pg/ml, p = 0.034). Based on ROC analysis, the threshold value that divides severe and non-severe was 78.6 pg/ml (AUC = 0.79). In the higher sBNP group (≥78.6 pg/ml), global end-diastolic volume index (GEDI) and intrathoracic blood volume index (ITVI), which indicate the cardiac preload, were significantly higher than in the low sBNP group. The systemic vascular resistance index (SVRI), the indicator for sympathetic activation and cardiac afterload, was also higher in the high BNP group. CONCLUSIONS: In severe SAH patients, sBNP elevation was significantly associated with the increase of both cardiac preload and afterload. sBNP may be a good severity biomarker in SAH patients, reflecting the systemic impact it makes on cardiovascular preload and afterload.

Corneal biomechanical properties measured by the ocular response analyzer in acromegalic patients.

PURPOSE: To investigate the effect of acromegaly on corneal biomechanical parameters. METHODS: This cross-sectional, comparative clinical study included 34 acromegalic patients and 30 age-matched and sex-matched healthy controls. Corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated and corneal-compensated intraocular pressure (IOPg and IOPcc, respectively) were measured using the Ocular Response Analyzer. Central corneal thickness (CCT) was determined with the ultrasonic pachymeter. RESULTS: The mean duration of disease for the acromegalic patients was 5.3 years. There was no significant difference between the groups regarding mean CH, CRF, IOPg and IOPcc values. The respective mean values in patients with acromegaly and controls were 10.3 ± 2.2 and 9.5 ± 1.5 mmHg (p = 0.13) for CH; 10.5 ± 2.4 and 9.7 ± 1.7 mmHg (p = 0.16) for CRF, 16.1 ± 3.6 and 15.5 ± 2.9 mmHg (p = 0.49) for IOPg, 16.8 ± 3.4 and 17.0 ± 2.8 mmHg (p = 0.82) for IOPcc, and 544.8 ± 32.2 and 530.7 ± 22.9 µm (p = 0.05) for CCT. A significant moderate correlation was detected between the duration of acromegaly and IOPg OD (r = 0.430, p = 0.01). There was no significant correlation between other ocular parameters and levels of GH and IGF-1 at the time of diagnosis, the status of control, adenoma type, radiotherapy treatment, and drug usage. CONCLUSIONS: In acromegalic patients, the duration of disease was correlated with IOPg OD level. Corneal biomechanical parameters and CCT values were not significantly different than those in age-matched and sex-matched healthy individuals.

Vitamin D in "early" primary Sjögren's syndrome: does it play a role in influencing disease phenotypes?

Beyond its well-established role in the maintenance of mineral homeostasis, 25-OH-vitamin D deficiency seems to be involved in the development and severity of several autoimmune diseases. To date, contrasting data have been reported regarding the presence of hypovitaminosis D in primary Sjögren's syndrome (pSS). To assess the prevalence of hypovitaminosis D in pSS at an early stage of the disease and to evaluate its impact on pSS clinical manifestations and disease activity, unselected consecutive subjects with recent onset dry mouth and/or dry eyes who underwent a comprehensive diagnostic algorithm for pSS (AECG criteria) were prospectively included in the study. The levels of 25[OH]-D3 were measured by monoclonal antibody immunoradiometric assay. Conditions of 25[OH]-D3 severe deficiency, deficiency, and insufficiency were defined as levels <10, <20, and 20-30 ng/ml, respectively, and their frequencies were investigated in pSS patients and controls. The levels of 25[OH]-D3 were also correlated with patients' demographic, clinical, and serologic features. Seventy-six consecutive females were included: 30/76 patients fulfilled the AECG criteria for pSS. The remaining 46/76 patients represented the control group. No statistical differences were found in the serum levels of 25[OH]-D3 between pSS patients [median levels = 20 ng/ml (IQR 9.3-26)] and controls [median levels = 22.5 ng/ml (IQR 15.6-33)]. In particular, the frequency of 25[OH]-D3 severe deficiency was not significantly different in patients with pSS when compared to controls (23 vs. 17.4 %, p value = 0.24). We found a significant correlation between serum 25[OH]-D3 levels and white blood cells count (r = 0.29, p = 0.01). More specifically, leukocytopenia was significantly associated with 25[OH]-D3 severe deficiency, being documented in 40 % of the subjects with a 25[OH]-D3 severe deficiency and in 11 % of the subjects without a severe vitamin D deficiency (p = 0.02). We did not observe any further association or correlation between hypovitaminosis D and pSS glandular and extra-glandular features. Although the role of hypovitaminosis D in pSS pathogenesis remains controversial, the results of this study encourage the assessment of vitamin D in specific pSS subsets that could mostly benefit from a supplementation.

Prospective investigation of anterior pituitary function in the acute phase and 12 months after pediatric traumatic brain injury.

PURPOSE: Although head trauma is common in childhood, there is no enough prospective study investigating both acute phase and 12 months after injury. Therefore, a prospective clinical trial was planned to evaluate the pituitary function in childhood in the acute and chronic phase after traumatic brain injury (TBI). METHODS: Forty-one children (27 boys and 14 girls, mean age 7 ± 4.3), who were admitted to neurosurgery intensive care unit due to head trauma, were included. Twenty-one (51.2 %) patients had mild, 10 (24.4 %) had moderate, and 10 (24.4 %) had severe TBI. Twenty-two of them were reevaluated 12 months after TBI. Basal pituitary hormone levels were measured during acute (first 24 h) and chronic phase of TBI. Additionally, in the chronic phase, GHRH-arginine test was used for the diagnosis of growth hormone (GH) deficiency. RESULTS: In the acute phase, 10 patients (24.4 %) had ACTH deficiency, and the overall 44.3 % of patients had at least one pituitary hormone dysfunction. All the pituitary hormone deficiencies during the acute phase were recovered after 12 months. Two patients (9.1 %) had new-onset GH deficiency in the chronic phase, and in one of them, ACTH deficiency was also present. CONCLUSIONS: Present prospective data clearly demonstrated that most of the hormonal changes in the early acute phase were transient, suggesting an adaptive response, and these changes did not predict the hormone deficiencies after 1 year. In the chronic phase, although GH deficiency was present, the frequency of TBI-induced hypopituitarism was clearly lower than the adult patients.

Change in growth differentiation factor 15 concentrations over time independently predicts mortality in community-dwelling elderly individuals.

BACKGROUND: Growth differentiation factor 15 (GDF-15) is emerging as a powerful risk indicator in both cardiovascular disease patients and community-dwelling individuals. We investigated GDF-15 concentrations and their changes over 5 years in elderly individuals from the community, together with the underlying conditions and prognostic implications of these measurements. METHODS: We analyzed GDF-15 concentrations using a sandwich immunoassay in participants from the PIVUS (Prospective Investigation of the Vasculature in Uppsala Seniors) study. Measurements were performed at both 70 (n = 1004) and 75 (n = 813) years of age. Median follow-up was 8.0 years. RESULTS: Over time, GDF-15 concentrations increased by 11.0% (P < 0.001). These changes were related to male sex, hypertension, diabetes, heart failure, renal function, and concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP). Significant relationships also emerged between changes in GDF-15 and changes in concentrations of NT-proBNP and C-reactive protein (CRP) and renal function between ages 70 and 75. The R(2) value of the model including all covariates was 0.20. GDF-15 concentrations independently predicted all-cause mortality [hazard ratio 4.0 (95% CI 2.7-6.0)] with results obtained at ages 70 and 75 as updated covariates. Baseline GDF-15 concentrations improved prognostic discrimination and reclassification [C statistic 0.06 (P = 0.006); integrated discrimination improvement = 0.030 (P = 0.004); category-free net reclassification improvement = 0.281 (P = 0.006)]. The change in GDF-15 concentrations over time independently predicted even all-cause mortality occurring after age 75 [hazard ratio 3.6 (95% CI 2.2-6.0)]. CONCLUSIONS: GDF-15 concentrations and their changes over time are powerful predictors of mortality in elderly community-dwelling individuals. GDF-15 concentrations increase with aging, and these changes are explained only partially by cardiovascular risk factors, indicators of neurohumoral activation and inflammation, and renal function. Thus GDF-15 reflects both cardiovascular and other biological processes closely related to longevity.

The utility of the IGF-I generation test in children with chronic kidney disease.

BACKGROUND: To determine if the insulin-like-growth factor (IGF-I) generation test is a marker for growth hormone (GH) sensitivity in children with chronic kidney disease (CKD). METHODS: This was a randomized cross-over study in which children with CKD received low-dose (0.025 mg/kg/day) and high-dose (0.05 mg/kg/day) GH therapy in the framework of a 7-day IGF-I generation test. Blood samples were collected on day 1 (D1; pre-dose) and on day 8 (D8; post 7 doses) of GH therapy. All subjects received GH for 12 months at 0.05 mg/kg/day. Serum IGF-I was measured by radioimmunometric assay. Normative historic data from healthy children and those with idiopathic short stature were used for comparison. RESULTS: Sixteen subjects (age 2-13 years) with creatinine clearances of between 25 and 75 ml/min/1.73 m(2) were enrolled. Annualized height velocity for all subjects was 10.3 ± 1.1 cm/year (mean ± standard deviation), with an annual change in height Z score of 0.7 ± 1.0. No correlation was found between the generated serum IGF-I levels (D8 - D1) and creatinine clearances, and with changes in height Z scores. Serum IGF-I levels on D1 and D8 in CKD subjects were lower than normative data, but with adequate IGF-I generation on D8. CONCLUSIONS: Children with CKD were able to respond to GH therapy with both growth and an increase in serum IGF-I levels, but the IGF-I generation test was not a good predictor of growth response in this cohort.

[Parathyroid hormone values in thyroid gland surgeries by harmonic scalpel and by conventional methods]. / Usporedba vrijednosti paratiroidnog hormona pri operacijama stitne zlijezde ultrazvucnim rezacem i konvencionalnim metodama.

We have examined if there are any differences in intraoperative and early postoperative concentrations of parathyroid hormone between the first group of patients, who had thyroidectomy surgery performed by harmonic scalpel, and the second group of patients operated on by standard techniqes with the use of electrocoagulation and ligature as primary hemostatic procedures. All the patients having total thyroidectomy had their blood taken in four measurement points; immediately after the induction anesthesia, 10 minutes after the first thyroid gland lobe removal, 10 minutes after total thyroid gland removal and 24 hours after the surgery. The blood samples were used to determine concentrations of the parathyroid hormone by an immunoradiometric test. The concentration comparison of parathyroid hormone between the first and the second group has not shown statistically significant difference for any of the four measurement points. The concentration comparison of parathyroid hormone within the same groups in relation to preoperational values (the first measurement point) has shown that in both groups the parathyroid hormone concentration, in all three post-incision measurement points, has been significantly lower in relation to the concentration measured before the surgery (p < 0.0005).

Limitations of Chromogranin A in clinical practice.

CONTEXT: Usefulness of circulating Chromogranin A (CgA) for the diagnosis of neuroendocrine tumors (NEN) is controversial. The aim of the present study was to assess the actual role of this marker as diagnostic tool. METHODS: Serum blood samples were obtained from 42 subjects affected with NEN, 120 subjects affected with non-endocrine neoplasias (non-NEN) and 100 non-neoplastic subjects affected with benign nodular goitre (NNG). Determination of CgA was performed by means of immunoradiometric assay. RESULTS: The CgA levels among NEN-patients were not significantly different from NNG and non-NEN subjects. The Receiver operating characteristic (ROC) curves analysis failed to identify a feasible cut-off value for the differential diagnosis between NEN and the other conditions. CONCLUSION: Serum CgA is not helpful for the first-line diagnosis of NEN.

Standardisation of a two-site PTH immunoradiometric assay using various solid phase formats.

BACKGROUND & OBJECTIVES: Estimation of parathyroid hormone (PTH) levels is important in the management of metabolic bone disorders. Here we describe a simple, sensitive and specific second generation immunoradiometric assay (IRMA) to detect intact PTH levels using different solid phase matrices. Different methods for immobilization of antibodies have also been evaluated. METHODS: Experiments were carried out with physical adsorption of antibodies, covalent coupling using 2 per cent glutaraldehyde and N,N`carbonyldiimidazole. In all cases, antibodies raised against C-terminal were used as solid phase agent. Detector antibodies were N terminal antibodies that were radio-iodinated with [125] I followed by gel purification. Several of the antibodies coupled to various solid phase matrices were incubated with PTH standards and the detector antibody as well as the commercially available tracer from DiaSorin kit to identify a suitable match pair. RESULTS: The best pair was polyclonal C-terminal PTH antibody along with the kit tracer from DiaSorin with regards to antibody coated to magnetic cellulose particles. Among the various antibodies and the solid phases evaluated, the best assay was obtained with the matched pair of antibodies (70×G67 and 70×G68) from Fitzgerald immobilized on polystyrene tubes. The polyclonal antibody against C-terminal PTH was chosen as the capture antibody and [125] I labelled polyclonal antibody against N-terminal PTH as the tracer. The sample values obtained in the antibody coated tubes were comparable to those obtained using a commercial kit. INTERPRETATION & CONCLUSIONS: The results indicated the feasibility of adopting this system for further development into a PTH IRMA for regular production as there is no indigenous kit available for intact PTH.

Development of a bioassay system for human growth hormone determination with close correlation to immunoassay.

Serum growth hormone (GH) level is measured largely through immunoassays in clinical practice. However, a few cases with bioinactive and immunoreactive GH have also been reported. We describe here a new bioassay system for GH determination using the BaF/GM cell line, which proliferates in a dose-dependent manner on hGH addition; cell proliferation was blocked by anti-hGH antibody. This bioassay had the lowest detection limit (∼0.02 ng/ml) reported thus far and the highest specificity for GH. The bioassay results were compared with those of an immunoradiometric assay across 163 patient samples in various endocrine states. A close correlation (the ratio of bioactivity/immunoreactivity was 1.04 ± 0.33, mean ± SD) was observed between bioactivity and immunoreactivity in these samples. The newly developed system is a specific, sensitive, easy, and fast bioassay system for GH determination; we consider it useful for evaluating GH bioactivity in various endocrine states.

Effects of hypothermia on NSE and S-100 protein levels in CSF in neonates following hypoxic/ischaemic brain damage.

AIM: The aim of the study was to evaluate the effects of hypothermia on neuron-specific enolase (NSE) and S-100 protein levels in cerebrospinal fluid (CSF) in neonates with hypoxic/ischaemic encephalopathy (HIE). METHODS: Fifty-one enrolled neonates with HIE were divided into two groups: hypothermia (n = 23) and control (n = 28). NSE and S-100 protein were measured with immunoradiometric assays. Amino acid neurotransmitters were also measured by reversed-phase high-performance liquid chromatography. Neurodevelopmental assessments were performed at 3 and 12 months of age. RESULTS: Neuron-specific enolase and S-100 levels were lower, and neurodevelopment outcome was better in the hypothermia group compared with the control group. Among the infants who received hypothermia, CSF NSE and S-100 were significantly higher in those who developed severe neurological impairment (mental development index or physical development index <70). There were no significant differences between the two groups in amino acid neurotransmitters. CONCLUSION: These results indicated that hypothermia was associated with decreased CSF NSE and S-100 level and correlated with neurodevelopmental outcome in infants with HIE.

Enhancing effect of human thyroid stimulating hormone (hTSH) monoclonal antibody in the hTSH immunoradiometric assay (IRMA) system.

Different monoclonal antibodies, both commercial and indigenously produced, were evaluated in various combinations to optimize an immunoradiometric assay (IRMA) system for human thyroid stimulating hormone (hTSH). During these studies, it was observed that mixing one of the indigenously produced hTSH monoclonal antibody (2B11) in the hTSH IRMA system using Immunotech (Beckman Coulter, Czech Republic) kit reagents, led to an overall increase in the assay binding and sensitivity (from 0.025 mIU/L to 0.015mIU/L) of this IRMA system. This is not a general property of all monoclonal antibodies against hTSH. The mechanism for this enhancement can be attributed to the formation of a multicomponent complex.